Cytotoxic Activity of LLO Y406A Is Targeted to the Plasma Membrane of Cancer Urothelial Cells

Abstract

Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.

Selective targeting of lectins and their macropinocytosis in urothelial tumours: translation from in vitro to ex vivo

“…It is known that endocytosis is diminished in vitro in highly differentiated NPU cells (Kreft et al 2009b ; Tratnjek et al 2017 ; Lojk et al 2018 ) and undifferentiated in NPU cells (Resnik et al 2021 ). To follow endocytosis of lectins in NPU cells, RT4 and T24 cells, live cells were incubated with FITC-conjugated lectins.…”

Proteome Analysis and In Vitro Antiviral, Anticancer and Antioxidant Capacities of the Aqueous Extracts of Lentinula edodes and Pleurotus ostreatus Edible Mushrooms

“…Our data show the expression of both ostreolysin and pleurotolysin in Pleurotus ostreatus. These are pore-forming proteins with highly selective anticancer activities [ 44 , 45 ]. Ostreolysin is a 15 kDa cytolytic protein with the ability to permeabilize erythrocytes and other cells at sub-micromolar concentrations.…”